Project Summary/Abstract The interferon (IFN)-based innate immune system is the first line of defense against viral infections and cancer. Under constant selective pressure, it has evolved to act quickly and accurately. Yet heightened alertness comes with a price?occasionally self molecules are improperly recognized as foreign and this leads to aberrant IFN production. Thus, the system exists in a delicate balance. From studying autoimmune disorders, we know that endogenous retroelements are a major source of ?foreign? nucleic acids and that they trigger aberrant IFN production when improperly controlled. But endogenous retroelements also play important beneficial roles in innate immunity. There's evidence that by maintaining tension in the system, they prime cells for effective antiviral responses. They also act as internal alarms to alert the immune system of danger when transcription is dysregulated in cancer. Several retroelement restriction factors are known and their mechanisms have been studied; however, there are likely more and there is much we don't know about how retroelements are controlled and how they affect innate immunity. The goals of this project are to identify novel host factors involved in retroelement control and to gain mechanistic insight into MOV10's mechanism of action. MOV10 is a potent retroelement restriction factor, and there is evidence that it may inhibit retroelements by a poorly understood small RNA mechanism. If true, identifying host factors required for MOV10 function could provide unique insights into the interplay between the protein-based IFN system used primarily by vertebrates and the more ancient small RNA-based mechanism of antiviral defense used by many other species. The approach that we'll take utilizes state-of-the-art CRISPR screening technology to knockout every gene in the human genome and identify host factors that restrict the replication of an intact LINE-1 retroelement. We will use this same approach to identify host proteins that are required for MOV10 function. The latter will provide useful mechanistic insights that we will explore in more detail using additional retroviral (HIV-1) and retroelement (Alu retrotransposon) tools. We expect that this work will provide new insights that will broaden our understanding of retroelement biology and innate immunity. These results may change the way we think about antiviral mechanisms, autoimmunity, and cancer.